Urinary tract infections (UTIs) are among the most common bacteriologic diseases, affecting 20% of women and costing $1 billion annually for diagnosis and treatment. Colonization of the vaginal mucosa by uropathogens is an early event in the pathogenesis of ascending UTIs. Vaginal fluid is the medium in which bacterial adhesins bind to epithelial cell receptors during colonization, but the influence of vaginal fluid on this interaction has received little attention. Recently, we discovered that type 1 piliated E. coli bound to glycoproteins in vaginal fluid and that binding was greater to fluid from colonized women than from noncolonized women (p<0.0001). Furthermore, vaginal fluid from colonized women generally enhanced adherence of E. coli to epithelial cells whereas fluid from noncolonized women did not alter adherence. By one- and two- dimensional (1D and 2D) gels, we discovered mannose containing glycoproteins that bound type 1 piliated E. coli. These data suggest that proteins in vaginal fluid modulate the interaction between bacteria and host cell surfaces and that qualitative changes in this fluid are associated with susceptibility to vaginal colonization and UTIs. Our goal is to identify and characterize specific glycoproteins that bind E. coli in vaginal fluid and which modulate vaginal mucosa colonization and susceptibility to UTIs. Three Specific Aims are proposed. 1. Test the hypothesis that the observed increases in receptivity of vaginal fluid for E. coli precede colonization of the vaginal mucosa with E. coli and UTIs. a. Changes in the binding capacity of vaginal fluid associated with vaginal colonization and UTIs will be identified by testing E coli adherence to vaginal fluid obtained sequentially from controls and patients, b. The effects of vaginal fluid on binding of E. coli to cultured epithelial cells will be tested. 2. Test the hypothesis that vaginal fluid specific glycoproteins that bind E. coli in vitro are associated with vaginal colonization and UTIs and these proteins differ in women who are colonized versus noncolonized with E. coli in vivo.a. The specific glycoproteins that bind E. coli will be identified and characterized by 1D and 2D gel electrophoresis. 3. Characterize bacteria binding proteins for N-terminal amino acid sequences and oligosaccharide components. a. Glycoproteins will be identified by N-terminal sequencing, b. Oligosaccharides of the glycoproteins will be characterized by fluorophore-assisted-carbohydrate- electrophoresis, mass spectrometry and sequential glycosidase treatments, c. Glycoproteins involved in E. coli binding will be quantitated by immunoassays. Results of this research will aid in the development of new diagnostic tests for measuring susceptibility to recurrent UTIs and in the elucidation of modifiers of bacterial colonization of the vaginal mucosa and prophylactic therapies for recurrent UTIs in women.